1. The primary cilia function as sensory antennae and play a critical signaling role in the development of multiple organs. Disruption of ciliary function underlies a variety of human diseases collectively known as ciliopathies. Sonic Hedgehog (SHH) signaling is one of the pathways mediated through primary cilia and regulates central nervous system development . FKBP8 null mutation in mice was previously shown to cause constitutive activation of SHH signaling in the neural tube leading to profound developmental defects, but the mechanism by which FKBP8 affects SHH signaling was unclear. We show that cilia morphogenesis in the neural tube and in fibroblasts of FKBP8 null mice remains intact. However, adenylyl cyclase III (AC3) fails to translocate to the ciliary membrane, thus abolishing cAMP synthesis and suppressing PKA activity. This leads to inhibition of the proteolytic processing of Gli transcription factors to the suppressor forms, which depends on PKA mediated phosphorylation. As generation of Gli repressors is quenched, SHH signaling in the cilia manifests constitutive activation, which explains the earlier observation in the FKBP null mutant mice. Using differentiated optic cups derived from FKBP8 null iPSCs, we were able to recapitulate this phenotype in photoreceptor connecting cilia. In summary, our study shows that the FKBP8 is essential for adenylate cycles to traffick into the cilia, and loss of FKBP8 disrupts ciliary signaling. (Philsang Hwang, in preparation). 2. Stem cell studies. We aim to make stem cell-derived photoreceptors that are well differentiated as indicated by the elaboration of outer segments. Such a culture system would be much better suited for retinal disease modeling, drug discoveries and provide donor tissues for transplantation. Given the dependence of photoreceptors on RPE, one of the critical components that is missing in current 3D retinal culture systems is a functioning RPE layer. Therefore, our long term goal is to develop a photoreceptor-RPE co-culture system. We have recently established a new aim, which is to differentiate iPSCs from Usher Type I patient into retinal organoids, and conduct cell biological studies to better understand the disease mechanism and to carry out drug screenings. In the first phase of the research, we have used an embryonic stem (ES) cell line (H9) to differentiate stem cells into RPE and after 60 days of induction we have been able to confirm successful differentiation of the ES cells into RPE cells. We have tested and compared RPE cells grown on conventional Trans-well system and on electrospun polymer support, which better simulates the physical properties of the Brunchs membrane, and have demonstrated the suitability of the latter as a substrate for growing and differentiating RPE from stem cells. Our differentiated RPE cells correctly display RPE markers, express typical proteins of mature RPE cells, have polarity of mature RPE cells, and display distinct RPE morphology such as: forming necessary tight junctions between the cells required for epithelial monolayer and hexagonal shape. This shows reliability and success of our protocol and allows a baseline comparison of differentiation of Induced Pluripotent Stem Cells (iPSCs). In the second phase which has recently begun, we are generating stem cell derived human and mouse retinal progenitor cells and testing different setups to achieve insulated culturing system where RPE and photoreceptor progenitors are in close juxtaposition with the correct polarity orientation and at the same time the media conditions on the RPE and photoreceptor side can be varied independently with no leakage (short circuiting) from the peripheries. Taking an reductionist approach, we are also testing liposome-mediated delivery of chromophore to retinal organoids, exposure to RPE apically and basally derived, conditioned media in an attempt to identify factors that facilitate outer segment formation (In progress. Ryan Kelley, John Wilson, in our Section in close collaboration with Dr. Swaroops Stem Cell group). 3. We have continued our study on protein post-translational modification in the cilia of photoreceptors and made significant new discoveries. The complex patterns of protein glutamylation contribute to the tubulin code that confers versatility to microtubule functions. Glutamylation is a form of post-translational modification initiated by the ligation of a single glutamate to the -carboxyl group of gene-encoded glutamate residues on target proteins known as monoglutamylation, and elongation of the branched peptide chains then generates polyglutamylation structures. Tubulin tyrosine ligase-like (TTLL) proteins are related enzymes that either initiate or elongate the glutamate side chains. Deglutamylases catalyzing the reverse reaction are members of the cytosolic carboxypeptidase (CCP) family, which similarly have preferential activities either to shorten long glutamate chains or remove the branching point glutamate. We show that cytosolic carboxypeptidase 5 (CCP5), the only CCP known to remove the branch point glutamate, is uniquely required for photoreceptor function and viability. In mice lacking CCP5, monoglutamylated tubulins are increased at the photoreceptor connecting cilia and axonemal microtubules but polyglutamylated tubulins remain unchanged. Photoreceptors degenerate soon after weaning with cones impacted more severely than rods. Prior to cell death there is marked ectopic accumulation of cone and rod opsins in the cell body. While increased tubulin monoglutamylation is seen systemically, mutant mice appear healthy other than retinal dystrophy and male infertility. These results are consistent with CCP5 being a critical deglutamulase responsible for removing the branch point glutamate in vivo, and support CCP5 mutations as a cause of human retinitis pigmentosa. The unexpected finding that an overt disease phenotype is restricted to retinal photoreceptors may be underscored by the exceptionally active transciliary trafficking of membrane receptors, and hence a greater sensitivity to changes in the tubulin code, relative to CNS neurons in general (Xun Sun et al., manuscript in preparation).